Recent studies have found that high temperatures cause oxidative stress and even mass mortality in Pacific oysters (Crassostrea gigas). The role of γ-aminobutyric acid (GABA) in improving antioxidative defense in aquatic animals is increasingly of interest. In the present study, the oxidative stress of Pacific oysters to high-temperature stress was examined, and the regulation of GABA on the antioxidative defense was further investigated. Following 6 h of exposure to 28 °C seawater, a significant increase in the mRNA expression levels of nuclear factor-E2-related factor 2 (Nrf2), superoxide dismutase (SOD), and catalase (CAT), as well as the activities of SOD and CAT, was observed in the gill, compared to those at 0 h. An increase of glutamate decarboxylase (GAD), GABA receptor (GABA_AR-α and GABA_BR-B) mRNA levels, and GABA contents were also detected after 28 °C exposure compared to those at 0 h. Furthermore, the activities and mRNA expression levels of SOD and CAT were significantly upregulated after GABA treatment, while decreased after either GAD inhibitor or GABA receptor inhibitor treatment under high-temperature stress. Meanwhile, the enhanced effects of GABA on antioxidant enzyme activities were reduced when Nrf2 was inhibited by ML385, accompanied by an increase in MDA content. After high-temperature stress, compared with the GABA treatment group, the activities and mRNA expression levels of SOD and CAT were significantly upregulated by GSK-3β inhibitor treatment. Meanwhile, the elevation of antioxidant enzyme activities by GABA was attenuated by the AKT inhibitor treatment. Collectively, GABA first activated GABA receptors under high-temperature stress and then increased the activities of SOD and CAT and reduced MDA content by AKT/GSK-3β and Nrf2 pathways to protect the oysters against oxidative damage upon stress. The present results offer new insights for understanding the regulation mechanisms of antioxidative defense by the neuroendocrine system in molluscs.